Design of a covalent protein-protein interaction inhibitor of SRPKs to suppress angiogenesis and invasion of cancer cells

Serine–arginine (SR) proteins are splicing factors that play essential roles in both constitutive and alternative pre-mRNA splicing. Phosphorylation of their C-terminal RS domains by SR protein kinases (SRPKs) regulates their localization and diverse cellular activities. Dysregulation of phosphorylation has been implicated in many human diseases, including cancers. Here, we report the development of a covalent protein–protein interaction inhibitor, C-DBS, that targets a lysine residue within the SRPK-specific docking groove to block the interaction and phosphorylation of the prototypic SR protein SRSF1. C-DBS exhibits high specificity and conjugation efficiency both in vitro and in cellulo. This self-cell-penetrating inhibitor attenuates the phosphorylation of endogenous SR proteins and subsequently inhibits the angiogenesis, migration, and invasion of cancer cells. These findings provide a new foundation for the development of covalent SRPK inhibitors for combatting diseases such as cancer and viral infections and overcoming the resistance encountered by ATP-competitive inhibitors.


Supplementary Figures
The crystal structure of SRPK1 in complex with a 7mer peptide (PDB ID: 7DD1).The peptide (orange) binds at the SRPK-specific docking groove.Inset: the lysine residues K602, K604 and K615 within the docking groove that serves as potential sites for proximity-enabled conjugation reaction are shown.The sidechain of E2 is not modeled due to lack of electron density.Based on the location of R3, E2 is expected to locate closely to K604 and K602.The distances between the amino groups of both lysines and the closest Ca of the 7mer are indicated.

Figure S3 .
Figure S3.Development of C-DBS.(a) Comparison of IC50 values of DBS1-1, DBS1-2, and DBS1-3.SRSF1 was phosphorylated by SRPK1△NS3 in the presence of different concentrations of the modified peptides using radioactive kinase activity assays.Radiolabelled phosphor-SRSF1 bands were quantified by ImageJ.DBS1-1 showed the best inhibitory effect

Figure
Figure S4.SRPK1 docking groove residues are important for the phosphorylation of substrate.(a) The binding affinity between SRPK1△NS3 and C-DBS was measured using MST.A constant concentration (50 nM) of His-SRPK1△NS3 was titrated with varying concentrations of C-DBS, yielding a Kd value of 1.5 μM.Data represent means ± SEM from three independent experiments.(b) SRPK1△NS3 or SRPK1△NS3_DM was used to phosphorylate GST-SRSF1.The activity assay was initiated by adding ATP and quenched by adding SDS loading buffer.Samples were resolved by Phos-tag SDS-PAGE and probed with anti-GST antibody.Nearly all SRSF1 was phosphorylated by SRPK1△NS3 and resulted in shifted bands.In contrast, most SRSF1 remained unphosphorylated in the presence of SRPK1△NS3_DM.

Figure
Figure S5.C-DBS specifically targets SRPKs (a) Structure alignment of the docking grooves of SRPK1 (green) and SRPK2 (pink).The two structures were superimposed using the α carbons of the kinases.The docking grooves of the two kinases are highly conserved, except that L568 in SRPK1 is replaced by H601 in SRPK2.K648 of SRPK2 corresponds to K604 of SRPK1.(b) C-DBS shows no inhibitory effect on other kinases.Related to Figure 4. Screening of the inhibitory effect of C-DBS (500 nM) against 140 protein kinases was performed by MRC PPU International Centre for Kinases Profiling, University of Dundee.C-DBS showed no obvious inhibition toward the kinases.Experiments were conducted in duplicate, and data were presented as means ± SEM.Abbreviations and assay conditions used for each kinase are defined at https://www.kinasescreen.mrc.ac.uk.

Figure
Figure S6.C-DBS is non-cytotoxic.Cell viability in several cell lines, including A549, MDA-MB-231, HeLa, and HEK-293 after C-DBS administration.Cells were treated with increasing concentrations of C-DBS for 24 hours before adding MTT.The absorbance at 570 nm was determined according to the manufacturer's instructions.C-DBS showed no cytotoxicity to these cell lines at concentrations up to 100 μM.Data represent means ± SEM from three independent experiments.

FigureFigure
Figure Figure S7.C-DBS inhibits the migration and invasion of MDA-MB-231 breast cancer cells.(a) C-DBS down-regulated phosphor-SR proteins in MDA-MB-231 cells in a dosedependent manner.MDA-MB-231 cells were treated with indicated concentrations of C-DBS and lysed with RIPA buffer supplemented with protease and phosphatase inhibitors.Total cell lysate was subjected to western blotting and phosphorylated SR proteins were probed with mAb104.The remaining p-SR levels versus β-tubulin were quantified using ImageJ.Data represent means ± SEM from three independent